Evolution of functional diversification of the paired box (Pax) DNA-binding domains

The Pax gene family consists of tissue-specific transcriptional regulators that always contain a highly conserved DNA-binding domain with six alpha-helices (paired domain), and, in many cases, a complete or residual homeodomain. Numerous genes of this family have been identified in animals, with the largest number found in vertebrates. Our evolutionary analyses indicate that the vertebrate Pax gene family consists of four well-defined and statistically supported groups: group I (Pax-1, 9), II (Pax-2, 5, 8), III (Pax-3, 7), and IV (Pax-4, 6). Group I paired domains share a most recent common ancestor with Drosophila Pox meso, group II with Pox neuro, group III with paired and gooseberry, and group IV with the eyeless gene. Two groups containing complete homeodomains (III and IV) are distantly related, and the intergroup relationships are (I,III), (II,IV). These four major groups arose before the divergence of Drosophila and vertebrates prior to the Cambrian radiation of triploblastic metazoan body plans. We conducted an analysis of fixed radical amino acid differences between groups in a phylogenetic context. We found that all four fixed radical amino acid differences between groups I and III are located exclusively in the N-terminal alpha-helices. Similarly, groups II and IV show three fixed radical differences in these alpha-helices but at positions different from those in groups I and III. Implications of such fixed amino acid differences in potentially generating sequence recognition specificities are discussed in the context of some recent experimental findings.